Summary: Poxviruses provide a unique system for studying the replication of DNA. Required enzymes and factors are encoded within the viral genome and DNA synthesis and processing occurs outside of the nucleus within the cytoplasmic compartment of the cell. Therefore, it has been possible to apply genetic and biochemical approaches to the study of DNA replication. Our effort has been directed towards ascertaining the structure and mode of replication of the poxvirus genome with particular emphasis placed on understanding the processing of the replicative intermediates. This project is endeavoring to discern the cis-acting and trans-acting components required for the processing of replicative intermediates, an integral process in vaccinia DNA replication. This knowledge will be used for the construction of highly attenuated safe poxvirus vectors and for the evaluation of presently used poxvirus vectors. The replication of vaccinia virus proceeds through concatemeric highly branched intermediates that are resolved into unit length DNA molecules. Mutational analysis has demonstrated that a cis-acting DNA sequence highly conserved among poxviruses as well as the palindromic structure of the concatemer is essential for resolution of the telomere and that resolution occurs by a process involving conservative strand exchange. A model for resolution involving site-specific recombination and oriented branch migration is consistent with this data. A separate, sequence-independent mechanism is responsible for the resolution of the numerous branch points present in the replicative intermediates. Our present efforts are directed towards determining the trans-acting protein components that participate in telomeric as well as branch resolution. We have recently concentrated on the identification and charaterization of a virion encoded nicking-joining enzyme. This protein is encoded at late times after infection, is included in the virion particle, and can cleave and cross-link DNA structures which mimic those found in poxvirus DNA replicative intermediates. We have identified the gene encoding for the activity, K4L, by genetic complementation. Recombinant viruses have been constructed in which the endogenous target gene has been replaced with an inducible copy of K4L. The protein encoded by K4L is not required for replication of vaccinia in tissue culture and its expression does not appear to affect the timing and distribution of the concatemeric intermediates.